Extraction of carotenoids from natural sources

ABSTRACT

A carotenoid-enriched fraction is extracted from natural sources, such as carrots, by (i) separating the carotenoid-containing natural source into a carotenoid-containing liquid fraction and a pulp fraction, (ii) adding a carotenoid precipitation agent including calcium chloride, calcium hydroxide, calcium lactate or calcium gluconate to the liquid fraction to form a carotenoid-enriched solid precipitate, and (iii) separating the carotenoid-enriched solid precipitate from the carotenoid-depleted liquid portion.

FIELD OF THE INVENTION

Broadly, the invention relates to the extraction of carotenoids fromcarotenoid-containing natural sources. Specifically, the inventionrelates to the extraction of carotenoids from a natural source, such ascarrots, by juicing the carrots, treating the juice with a carotenoidprecipitation agent including calcium chloride, calcium hydroxide,calcium lactate or calcium gluconate, to produce a carotenoid-enrichedsolid precipitate portion and a carotenoid-depleted liquid portion, andseparating the solid and liquid portions.

BACKGROUND

Carotenoids are a class of naturally occurring pigments found in traceamounts in the tissues of higher plants, algae, bacteria and fungi.Carotenoids are polyenes having a C₄₀ carbon skeleton (phytoene) whichcontains an extended network of single and double bonds. The variouscarotenoids are formed by chemically modifying this C₄₀ carbon skeleton.For example, dehydrogenation of phytoene yields the carotenoid lycopenewhich is responsible for the color of tomatoes and cyclization of bothends of lycopene yields the carotenoid β-carotene which is responsiblefor the color of carrots.

Carotenoids, such as β-carotene, are valuable pigments useful forcoloring various comestibles, such as margarine, as they avoid thehealth concerns associated with synthetic pigments and actually possesssignificant nutritional value (β-carotene is a precursor to theformation of retinal and vitamin A in humans).

Because carotenoids occur naturally in only trace amounts, thecarotenoids must be extracted in concentrated form in order to beuseful. Ordinarily, carotenoids are extracted from natural sources bytreating the material with a carotenoid-solubilizing hydrocarbonsolvent, such as hexane, or chloroform, separating thecarotenoid-containing hydrocarbon solvent from the remainder of thematerial, and then driving off the hydrocarbon solvent to produce acarotenoid-enriched solid product.

In addition to carotenoids, plants contain a variety of otherconstituents which are soluble in hydrocarbon solvents such as variousproteins and lipids. Accordingly, the carotenoid-enriched solid producttypically includes significant amounts of other components in additionto the carotenoid(s).

Use of a hydrocarbon solvent to extract the carotenoids significantlyincreases the cost and complexity of the extraction procedure due to thecost of the hydrocarbon solvent, the cost of removing the hydrocarbonsolvent from the final product, the cost of recovering the removedhydrocarbon solvent, and the cost of disposing of contaminatedhydrocarbon solvent which cannot be reused. In addition, use of ahydrocarbon solvent to effect extraction of carotenoids results insignificant environmental damage due to the release of hydrocarbon fumesinto the atmosphere and the need to dispose of contaminated hydrocarbonsolvent which cannot be reused.

Accordingly, a substantial need exists for a simple and environmentallysafe process of extracting carotenoids from carotenoid-containingnatural sources which avoids the need to utilize a hydrocarbon solvent.

SUMMARY

We have discovered a process for extracting carotenoids fromcarotenoid-containing natural sources, such as carrot juice, whichincludes the steps of (i) separating the carotenoid-containing naturalsource into a carotenoid-containing liquid fraction and a pulp fraction,(ii) contacting the liquid fraction with an effective fractionatingamount of a carotenoid precipitation agent including calcium chloride,calcium hydroxide, calcium lactate or calcium gluconate, so as tofractionate the liquid fraction into a carotenoid-enriched solidprecipitate portion and a carotenoid-depleted liquid portion, and (iii)separating the carotenoid-enriched solid portion from thecarotenoid-depleted liquid portion.

The carotenoid-enriched solid fraction may be utilized directly or maybe further purified so as to separate the carotenoid(s) from the otherconstituents in the solid fraction by any suitable separation technique.Preferred techniques including chemical and enzymatic hydrolysis andchemical and enzymatic degradation whereby the noncarotenoidconstituents are rendered separable from the carotenoid(s) in an aqueousmedia. While the carotenoid-enriched solid fraction may be purifiedutilizing conventional organic liquid or solid phase extraction, use ofsuch an extraction procedure destroys the desired organic solventlessnature of the process and therefore is not favored.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph obtained from the data in Table One indicating theconcentration of carotene recovered from treated and untreated carrotjuice.

FIG. 2 is a bar graph indicating the degree of carotenoid separationachieved by the addition of from 0.5 to 2 wt % of various salts to wholecarrot juice with the highest degree of separation obtained for eachsalt within the tested concentration range reported on the graph.

FIG. 3 is a bar graph indicating the degree of carotenoid separationachieved by the addition of various concentrations of calcium salts towhole carrot juice.

FIG. 4 is a bar graph indicating the speed of carotenoid separationachieved by the addition of calcium chloride to whole carrot juice atvarious pH levels.

DETAILED DESCRIPTION OF THE INVENTION INCLUDING A BEST MODE

A carotenoid-enriched solid product may be simply, quickly andefficiently extracted from carotenoid-containing natural sources, suchas carrots, without the use of a hydrocarbon solvent by (i) separatingthe carotenoid-containing natural source into a carotenoid-containingliquid fraction and a pulp fraction, (ii) treating thecarotenoid-containing liquid fraction with a carotenoid precipitationagent including calcium chloride, calcium hydroxide, calcium lactate orcalcium gluconate, so as to fractionate the liquid fraction into acarotenoid-enriched solid precipitate portion and a carotenoid-depletedliquid portion, and (iii) separating the liquid and solid portions byconventional means.

We believe that substantially any carotenoid-containing natural sourcemay be effectively fractionated in accordance with the invention toproduce a carotenoid-enriched product including specifically, but notexclusively, fruits such as pineapples and oranges; vegetables such ascarrots, spinach, sweetpotatoes and tomatoes; algae such as DunaliellaSalina; bacteria such as those of the order Mucorales including C.trispora and Blakeslea Circinans; and fungi. Based upon ease ofavailability, low cost, and high concentration of commercially valuableβ-carotene, the starting material of preference is carrots.

The first step in the process of the invention is to separate thecarotenoid-containing natural source into a carotenoid-containing liquidfraction and a pulp fraction. While the exact mechanism employed toachieve this separation depends upon several factors, including thespecific carotenoid source, such separation can typically be achieved bysimply juicing the carotenoid source and filtering the juice through acourse-mesh filter. Disruption of the cell structure of the carotenoidsource during separation generally inherently results in transfer of thecarotenoid(s) in the carotenoid source from the pulp fraction to theliquid fraction.

Addition of a carotenoid precipitation agent including calcium chloride,calcium hydroxide, calcium lactate or calcium gluconate, to thecarotenoid-containing liquid fraction causes precipitation of acarotenoid-enriched fraction which may be separated from the remainingcarotenoid-depleted liquid fraction by conventional separation methods.

The physical and/or chemical mechanism(s) responsible for suchprecipitation of a carotenoid-enriched solid fraction by the addition ofa source of ionizable calcium is not fully understood. In an effort toascertain whether the pectin and/or proteins contained in the liquidfraction participate in this phenomena, samples of carrot juice weretreated with a protease enzyme and a pectinase enzyme prior to additionof the carotenoid precipitation agent calcium chloride (See Table Threeand accompanying conclusions). Such pretreatment with an enzyme todegrade the proteins (protease) and pectin (pectinase) contained in thejuice resulted in no noticeable alteration in fractionation of the juiceby the calcium chloride. Accordingly, it appears that the proteins andpectin contained in the carotenoid-containing liquid fraction do notplay an independently active role in the physical and/or chemicalmechanism responsible for precipitation of a carotenoid-enriched solidfraction from the liquid fraction by the addition of the listedcarotenoid precipitation agents.

Efforts to obtain comparable fractionation with other mineral salts suchas sodium chloride, potassium chloride, magnesium chloride, calciumcarbonate, and calcium phosphate; and caustics such as sodium hydroxideand potassium hydroxide have proven substantially unsuccessful (See FIG.2 and Protocol-salt treatment). Accordingly, it appears that the onlyreagents capable of providing effective fractionation are those capableof providing ionized calcium under those conditions present in thejuice, such as calcium chloride, calcium hydroxide, calcium lactate andcalcium gluconate, with calcium chloride appearing to providesignificantly better separation at lower concentrations.

Referring to FIG. 3, superior separation of the carotenoids into adistinct solid precipitate portion may be achieved from acarotenoid-containing liquid fraction of a carotenoid-containing naturalsource by adding calcium chloride at concentrations of from about 0.01to 10 wt %, preferably about 0.05 to 3 wt %, calcium hydroxide atconcentrations of from 0.01 to 10 wt %, preferably about 0.05 to 3 wt %,calcium lactate at concentrations of from about 2 to 10 wt %, preferablyabout 2 to 4 wt %, and calcium gluconate at concentrations of from about4 to 10 wt %, preferably about 4 to 6 wt %, with the best overallseparation achieved with calcium chloride.

Referring to FIG. 1 and Table One, the liquid fraction may besignificantly fractionated by heating the liquid fraction totemperatures of at least 60° C.

Separation of the liquid fraction by treating with the listed carotenoidprecipitation agents in accordance with the process of the invention maybe achieved under ambient conditions. However, for the purpose ofincreasing the rate of separation, the liquid fraction is preferablyheated to a temperature above about 40° C. and preferably between about40° to 60° C. while being treated with one of the listed carotenoidprecipitation agents.

Effective fractionation may be obtained by treating the liquid fractionwith one of the listed carotenoid precipitation agents for as little asabout one minute (See Table Two and accompanying comments). Whileseveral factors may affect the optimum contact period such as thespecific carotenoid precipitation agent employed, the concentration ofthe carotenoid precipitation agent, the temperature of the liquidfraction, and the type of carotenoid-containing source, effectivefractionation generally occurs in less than one hour, and morespecifically in less than thirty minutes. Generally, optimumfractionation appears to be achieved for carrot juice heated to slightlyelevated temperatures and treated with one of the listed carotenoidprecipitation agents at a contact period of about five to ten minutes.Contact periods of less than about five minutes result in a slightlyless effective separation while contact periods of greater than aboutten minutes produce little additional separation.

It is believed that optimal fractionation may also be obtained bytreating the liquid fraction with one of the listed carotenoidprecipitation agents for less than about five minutes, possibly lessthan about one minute, by employing temperatures between about 80°-120°C.

Referring to FIG. 4 and Table Four, the pH of the liquid fraction canaffect the rate of separation and overall separation efficiency. Carrotjuice has a natural pH of about 6.0. Carrot juice having a pH adjustedwith NaOH as necessary to between about 6 to 7 appears to provide themost complete separation (clearer liquid fraction) while that adjustedto between about 10 to 11 appears to provide nearly instantaneousseparation after addition of the carotenoid precipitation agent.

Separation of the carotenoid-enriched solid precipitate portion from thecarotenoid-depleted liquid portion may be achieved by any conventionalmethod, including centrifugation/decantation/freeze-drying,centrifugation/decantation/heat drying, heat drying, evaporation, andthe like.

The solid carotenoid-enriched portion may be utilized without furtherprocessing wherever the pigmentation provided by the concentratedcarotenoid(s) is desired. If desired, the carotenoid-enriched solidportion may be further refined to separate the carotenoid(s) from theother precipitated components such as ash, carbohydrates, lipids, andproteins, and obtain a more concentrated carotenoid-containing productby employing conventional purification techniques such as chemical andenzymatic hydrolysis, chemical and enzymatic degradation, liquid-liquidextraction, solid phase extraction, etc.

COMPARATIVE TESTING PROTOCOL

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into three40 ml samples and placed into 125 ml Erlenmeyer flasks. A first of thesamples was left untreated. A second of the samples was heated bysubmerging the sample into a waterbath maintained at 60° C. for twentyminutes. A third of the samples was treated with calcium chloridedihydrate and then heated by submerging the sample into a waterbathmaintained at 60° C. for twenty minutes.

The samples were centrifuged for 15 minutes at 2000×g to produce a solidpellet and a liquid supernatant. The supernatant was decanted from thesolid pellet and the pellet freeze-dried. The freeze-dried pellets wereanalyzed for α and β-carotene concentrations using HPLC technology inaccordance with the HPLC Protocol set forth below.

TESTING PROTOCOL HPLC

Freeze-dried pellets were pulverized with a mortar and pestle.Approximately 0.025 grams of each sample were added to 4 milliliters ofwater. This mixture was then extracted with three 10 milliliter portionsof a combination of petroleum ether:acetone (50:50 v/v) and filteredthrough a Buchner funnel. The filer cake was discarded and the filtrateevaporated to dryness under nitrogen. The resulting dried extract wasresuspended in 4 milliliters of pure chloroform. A 1 milliliter sampleof the resuspended extract was diluted with 4 milliliters of chloroform.The diluted extract was filtered through a 0.45 μm filter and thenanalyzed by high performance liquid chromotography (HPLC) in accordancewith the procedure set forth in Journal of Food Science, Vol 52, No. 3,pp. 744-46, 1987 for α and β-carotene content.

Sample carotene content was quantified by comparison of peak areas withpeak areas of authentic standards of known concentration obtained fromSigma Chemical Co. Standard concentrations used were 12.5, 25.0, 50.0,and 100.0 ug/ml.

The milligrams of α and β carotene, per gram of dried solid material areset forth in Table One and FIG. 1.

TESTING PROTOCOL Salt Treatment with Various Salts

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. Individual sampleswere treated with a salt selected from sodium chloride, potassiumhydroxide, magnesium chloride hexahydrate, calcium chloride dihydrate,potassium chloride, sodium hydroxide, calcium carbonate, calciumphosphate, calcium hydroxide, calcium lactate and calcium gluconate atconcentrations of 0.5, 1.0 and 2.0 wt % for sixty minutes. The extent ofseparation was observed and recorded in accordance with the nomenclatureset forth below.

    ______________________________________                                        Nomenclature                                                                  ______________________________________                                        0                No separation                                                1                Slight separation                                            2                Fair separation                                              3                Good separation                                              4                Excellent separation                                         ______________________________________                                    

The best degree of separation attained for each of the salts, at thetested concentrations, is plotted in FIG. 2.

The treated samples were centrifuged for 15 minutes at 20° C. and 2000×gto produce a solid pellet and a liquid supernatant. The supernatant wasdecanted from the solid pellet and the pellet freeze-dried.

TESTING PROTOCOL Salt Treatment with Calcium Salts

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. Duplicate samples weretreated with a calcium salt selected from calcium chloride dihydrate,calcium carbonate, calcium phosphate, calcium hydroxide, calcium lactateand calcium gluconate at concentrations of 1, 2 and 4 mmoles for sixtyminutes. The extent of separation was observed and recorded inaccordance with the nomenclature set forth below.

    ______________________________________                                        Nomenclature                                                                  ______________________________________                                        0                No separation                                                1                Slight separation                                            2                Fair separation                                              3                Good separation                                              4                Excellent separation                                         ______________________________________                                    

The degree of separation attained for each of the calcium salts at eachconcentration level is plotted in FIG. 3.

The treated samples were centrifuged for 15 minutes at 25° C. and 2000×gto produce a solid pellet and a liquid supernatant. The supernatant wasdecanted from the solid pellet and the pellet freeze-dried.

TESTING PROTOCOL Heating and Salt Treatment

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. The samples weretreated with a salt of the type (Salt-type) and in the amount(Salt-grams) set forth in Table Two and immersed in a constanttemperature water bath heated to 60° C. for the period of time (ContactTime) set forth in Table Two. The extent of separation was observed andrecorded in accordance with the nomenclature set forth below.

    ______________________________________                                        Nomenclature                                                                  ______________________________________                                        0                No separation                                                1                Slight separation                                            2                Fair separation                                              3                Good separation                                              4                Excellent separation                                         ______________________________________                                    

The treated samples were centrifuged for 15 minutes at 25° C. and 2000×g to produce a solid pellet and a liquid supernatant. The supernatantwas decanted from the solid pellet and the pellet freeze-dried.

TESTING PROTOCOL Heating, Salt and Enzyme Treatment

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. The samples weretreated with an enzyme of the type (Enzyme-type) and in the amount(Enzyme-grams) set forth in Table Three. The enzyme containing samplewas immersed in a constant temperature water bath heated to thetemperature set forth in Table Three (Bath Temp) for the period of timeset forth in Table Three (Contact Time Heat+Enzym). The enzyme./heattreated samples were treated with a salt of the type (Salt-type) and inthe amount (Salt-grams) set forth in Table Three for the period of time(Contact Time-Salt) set forth in Table Three. The extent of separationwas observed and recorded in accordance with the nomenclature set forthbelow.

    ______________________________________                                        Nomenclature                                                                  ______________________________________                                        0                No separation                                                1                Slight separation                                            2                Fair separation                                              3                Good separation                                              4                Excellent separation                                         ______________________________________                                    

The treated samples were centrifuged for 15 minutes at 25° C. and 2000×gto produce a solid pellet and a liquid supernatant. The supernatant wasdecanted from the solid pellet and the pellet freeze-dried.

TESTING PROTOCOL CaCl and pH Treatment

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. The pH of the sampleswere adjusted to 8, 9, 10 or 11 with a solution of 10N NaOH and thentreated with 0.294 g of calcium chloride dihydrate for thirty minutes.Excellent separation was observed for all samples. The length of timerequired to achieve excellent separation after addition of the Calciumchloride was recorded.

    ______________________________________                                        Nomenclature                                                                  ______________________________________                                        0                No separation                                                1                Slight separation                                            2                Fair separation                                              3                Good separation                                              4                Excellent separation                                         ______________________________________                                    

The treated samples were centrifuged for 15 minutes at 25° C. and 2000×gto produce a solid pellet and a liquid supernatant. The supernatant wasdecanted from the solid pellet and the pellet freeze-dried.

TESTING PROTOCOL Heating, Salt and pH Treatment

Carrots were juiced with a disintegrator and the juice separated fromthe pulp by centrifugation or pressing. The juice was divided into 40 mlsamples and placed into 125 ml Erlenmeyer flasks. The pH of the sampleswere adjusted as set forth in Table Four with a solution of 10N NaOH,treated with 0.294 g of calcium chloride dihydrate, and then immersed ina constant temperature water bath heated to 60° C. for ten minutes.Excellent separation was observed for all samples. The extent ofseparation was observed and recorded in accordance with the nomenclatureset forth below.

The treated samples were centrifuged for 15 minutes at 25° C. and 2000×gto produce a solid pellet and a liquid supernatant. The supernatant wasdecanted from the solid pellet and the pellet freeze-dried.

                                      TABLE ONE                                   __________________________________________________________________________    Comparison of Carotene Concentrations                                         Heat Treated/Calcium Treated/Untreated Carotene                                                Concentration                                                   Bath                                                                              Contact        α                                                                            β                                                                             Total                                            Temp                                                                              Time in                                                                             Salt                                                                              Salt (mg/g)                                                                             (mg/g)                                                                             (mg/g)                                        #  (°C.)                                                                      Bath (min)                                                                          (type)                                                                            (mmoles)                                                                           [mg/L]                                                                             [mg/L]                                                                             [mg/L]                                        __________________________________________________________________________    100                                                                              --  --    --  --   (0.679)                                                                            (0.683)                                                                            (1.362)                                                             [72.0]                                                                             [72.5]                                                                             [144.5]                                       101                                                                              60  20    --  --   (3.73)                                                                             (6.40)                                                                             (10.13)                                                             [46.75]                                                                            [80.0]                                                                             [126.75]                                      102                                                                              60  20    CaCl.sub.2                                                                        2    (4.48)                                                                             (7.96)                                                                             (12.44)                                                             [56.0]                                                                             [99.5]                                                                             [155.5]                                       __________________________________________________________________________     Observation: Both heattreatment and calciumtreatment of carrot juice          produce a coagulum that is highly enriched in α and β carotene     Most of the α and β carotene in the whole juice is recovered b     these processes. Heat treatment combined with calcium treatment appears t     increase the total amount of carotene (α  and β) recovered by      about 23% over that recovered by heat treatment only.                    

                  TABLE TWO                                                       ______________________________________                                        Heat + Inorganic Salts                                                        ______________________________________                                                Contact   Salt       Salt                                             #       Time (min)                                                                              (type)     (grams)                                                                              Separtn                                   ______________________________________                                        200a    20        CaCl.sub.2 (0.294)                                                                              4                                         200b    20        CaCl.sub.2 (0.294)                                                                              4                                         201a    20        Ca(OH).sub.2                                                                             (0.148)                                                                              4                                         201b    20        Ca(OH).sub.2                                                                             (0.148)                                                                              4                                         Cntrl   10        --         --     4                                         Cntrl   10        --         --     4                                         Cntrl   20        --         --     4                                         Cntrl   20        --         --     4                                         210a    05        CaCl.sub.2 (0.147)                                                                              4                                         210b    05        CaCl.sub.2 (0.147)                                                                              4                                         211a    10        CaCl.sub.2 (0.147)                                                                              4                                         211b    10        CaCl.sub.2 (0.147)                                                                              4                                         212a    20        CaCl.sub.2 (0.147)                                                                              4                                         212b    20        CaCl.sub.2 (0.147)                                                                              4                                         213a    05        CaCl.sub.2 (0.294)                                                                              4                                         213b    05        CaCl.sub.2 (0.294)                                                                              4                                         214a    10        CaCl.sub.2 (0.294)                                                                              4                                         214b    10        CaCl.sub.2 (0.294)                                                                              4                                         215a    20        CaCl.sub.2 (0.294)                                                                              4                                         215b    20        CaCl.sub.2 (0.544)                                                                              4                                         216a    05        CaCl.sub.2 (0.544)                                                                              4                                         216b    05        CaCl.sub.2 (0.544)                                                                              4                                         217a    10        CaCl.sub.2 (0.544)                                                                              4                                         217b    10        CaCl.sub.2 (0.544)                                                                              4                                         218a    20        CaCl.sub.2 (0.544)                                                                              4                                         218b    20        CaCl.sub.2 (0.544)                                                                              4                                         ______________________________________                                        Trial          #Comments                                                      ______________________________________                                        200a/b         Separation was excellent.                                                     Supernatant very clear and                                                    pellets very firm.                                                            Decantation was very easy.                                     201a/b         Separation was excellent.                                                     Pellets were very firm.                                                       Decantation was very easy.                                     ______________________________________                                    

                                      TABLE THREE                                 __________________________________________________________________________    Heat + Inorganic Salts + Enzymes                                                 Bath                                                                              Time     Time                                                             Temp                                                                              (Heat + Enzyme)                                                                        (Salt)                                                                            Salt                                                                              Salt Enzyme                                                                             Enzyme                                      #  (°C.)                                                                      (hrs)    (hrs)                                                                             (type                                                                             (grams)                                                                            (type)                                                                             (grams)                                                                            Separtn                                __________________________________________________________________________    300a                                                                             40°                                                                        .sup. 05.sup.bb                                                                        05.sup.c                                                                          CaCl.sub.2                                                                        (0.4)                                                                              Protease                                                                           (0.004)                                                                            4                                      300b                                                                             40°                                                                        .sup. 05.sup.bb                                                                        05.sup.c                                                                          CaCl.sub.2                                                                        (0.4)                                                                              Protease                                                                           (0.004)                                                                            4                                      310a                                                                             30°                                                                        18.sup.b .sup. 01.sup.cc                                                                   CaCl.sub.2                                                                        (0.147)                                                                            Pectinase                                                                          (1 ml)                                                                             4                                      310b                                                                             30°                                                                        18.sup.b .sup. 01.sup.cc                                                                   CaCl.sub.2                                                                        (0.147)                                                                            Pectinase                                                                          (1 ml)                                                                             4                                      __________________________________________________________________________

Observations

Treatment of a 40 ml sample of whole carrot juice with 0.01 wt %protease (incubation at 40° C. for 5 hours) produced no visibleseparation. Subsequent addition of 0.0027 moles of Ca⁺⁺ in the form ofCaCl₂.2H₂ O produced rapid separation resulting in a clearer supernatantmoiety and a lower viscosity carotenoid moiety than typically achieved.This indicates that degradation of the protein present in whole carrotjuice does not effect separation of carotenoids when used along but canassist in achieving greater separation when used in conjunction withcalcium treatment.

Treatment of a 40 ml sample of whole carrot juice with 0.01 wt %pectinase (incubation at 30° C. for 18 hours) produced no visibleseparation. Subsequent addition of 0.001 moles of Ca⁺⁺ in the form ofCaCl₂.2H₂ O initiated separation within three minutes resulting in aclear supernatant moiety and a well defined carotenoid moiety within onehour. This indicates that degradation of the pectin present in wholecarrot juice does not effect separation of carotenoids when used alongbut can assist in hastening separation when used in conjunction withcalcium treatment.

                  TABLE FOUR                                                      ______________________________________                                        Heat + Inorganic Salt w/pH Adjustment                                                        Bath    Contact                                                      pH       Temp    Time   Salt  Salt   Se-                                #     Reading  (°C.)                                                                          (min)  (type)                                                                              (grams)                                                                              partn                              ______________________________________                                        Cntrl --       --      --     --    --     0                                  Cntrl --       --      --     --    --     0                                  400a  6.50     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  400b  6.50     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  401a  7.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  401b  7.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  402a  8.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  402b  8.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  403a  9.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  403b  9.00     60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  404a  10.00    60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  404b  10.00    60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  405a  11.00    60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  405b  11.00    60°                                                                            10     CaCl.sub.2                                                                          (0.294)                                                                              4                                  ______________________________________                                         .sup.b Sample under constant agitation at 87 cycles/min during immersion      in water bath.                                                                .sup. bb Sample under constant agitation at 85 cycles/min during immersio     in water bath.                                                                .sup.c Sequence: Enzyme and Salt added to sample with subsequent heat         treatment.                                                                    .sup.cc Sequence: Enzyme and Salt added with period of heat treatment         occuring inbetween.                                                      

Trial #s with same tens diget (110s, 120s, etc.) indicate that trialswere conducted employing same source of carrot juice. Trial #s differingonly as to designation (a) (b) indicates pair of identically treatedsamples.

The specification, including the examples, is intended to aid in acomplete and unlimited understanding of the invention. Since variousembodiments of the invention may be made without departing from thespirit and scope of the invention, the scope of the invention lies inthe claims hereinafter appended.

I claim:
 1. A process for extracting carotenoids from acarotenoid-containing natural source which comprises the steps of:(a)separating a natural source of a carotenoid into a carotenoid-containingliquid fraction and a pulp fraction, (b) contacting thecarotenoid-containing liquid fraction with an effective fractionatingamount of calcium chloride so as to fractionate the liquid fraction intoa carotenoid-enriched solid precipitate portion and acarotenoid-depleted liquid portion, and (c) separating thecarotenoid-enriched solid portion from the carotenoid-depleted liquidportion.
 2. The process of claim 1 wherein the natural source iscarrots.
 3. The process of claim 1 wherein the liquid fraction iscontacted with about 0.01 to about 10 wt % calcium chloride.
 4. Theprocess of claim 1 wherein the liquid fraction is contacted with about0.05 to about 3 wt % calcium chloride.
 5. The process of claim 1 whereinthe liquid fraction is contacted with the calcium chloride under ambientconditions.
 6. The process of claim 1 wherein the liquid fraction iscontacted with the calcium chloride at a temperature above about 40° C.7. The process of claim 1 wherein the liquid fraction is contacted withthe calcium chloride at a temperature of about 40° to 60° C.
 8. Theprocess of claim 7 wherein the liquid fraction is contacted with thecalcium chloride for a time period of at least about 10 minutes.
 9. Theprocess of claim 8 wherein the liquid fraction is contacted with thecalcium chloride for a time period of about 10 to 30 minutes.
 10. Theprocess of claim 1 wherein the pH of the liquid fraction is betweenabout 6 to
 8. 11. A process for extracting carotenoids from acarotenoid-containing natural source without the use of a hydrocarbonsolvent, which comprises the steps of:(a) separating a natural source ofa carotenoid into a carotenoid-containing liquid fraction and a pulpfraction, (b) contacting the liquid fraction with a hydrocarbon solventfree precipitating agent selected from the group consisting of calciumlactate and calcium gluconate wherein about 2 to 10 wt % precipitatingagent is used when the precipitating agent is calcium lactate and about4 to 10 wt % precipitating agent is used when the precipitating agent iscalcium gluconate; the precipitating agent effective for fractionatingthe liquid fraction into a carotenoid-enriched solid precipitate portionand a carotenoid-depleted liquid portion wherein both the solid andliquid are free of hydrocarbon solvent, and (c) separating thecarotenoid-enriched solid portion from the carotenoid-depleted liquidportion without the use of a hydrocarbon solvent so as to form acarotenoid-enriched solid extract which has been contacted with ahydrocarbon solvent during extraction.
 12. The process of claim 11wherein the precipitating agent is calcium lactate or calcium gluconateand the liquid fraction is contacted with about 2 to 4 wt %precipitating agent when the precipitating agent is calcium lactate andabout 4 to 6 wt % precipitating agent when the precipitating agent iscalcium gluconate.
 13. The process of claim 11 wherein the liquidfraction is contacted with the precipitating agent under ambientconditions.
 14. A process for extracting carotenoid from acarotenoid-containing natural source without the use of a hydrocarbonsolvent, which comprises the steps of:(a) separating a natural source ofa carotenoid into a carotenoid-containing liquid fraction and a pulpfraction, (b) contacting the liquid fraction with an effectivefractionating amount of a hydrocarbon solvent free precipitating agentselected from the group consisting of calcium chloride, calcium lactateand calcium gluconate at a temperature of about 40° to 60° C. and for atime period of less than about 1 hour so as to fractionate the liquidfraction into a carotenoid-enriched solid precipitate portion and acarotenoid-depleted liquid portion wherein both the solid and liquidportions are free of hydrocarbon solvent, and (c) separating thecarotenoid-enriched solid portion from the carotenoid-depleted liquidportion without the use of a hydrocarbon solvent so as to form acarotenoid-enriched solid extract which has not been contacted with ahydrocarbon solvent during extraction.
 15. The process of claim 14wherein the liquid fraction is contacted with the precipitating agentfor a time period of less than about 30 minutes.
 16. The process ofclaim 14 wherein the liquid fraction is contacted with the precipitatingagent for a time period of less than about 20 minutes.
 17. The processof claim 14 wherein the liquid fraction is contacted with theprecipitating agent for a time period of less than about 10 minutes. 18.The process of claim 14 wherein the liquid fraction is contacted withthe precipitating agent for a time period of less than about 5 minutes.19. The process of claim 14 wherein the liquid fraction is contactedwith the precipitating agent for a time period of less than about 2minutes.
 20. The process of claim 14 wherein the liquid fraction iscontacted with the precipitating agent for a time period of less thanabout 1 minute.
 21. A process for extracting carotenoids from acarotenoid-containing natural source which comprises the steps of:(a)separating a natural source of a carotenoid into a carotenoid-containingliquid fraction and a pulp fraction. (b) contacting thecarotenoid-containing liquid fraction with an effective fractionatingamount of calcium lactate so as to fractionate the liquid fraction intoa carotenoid-enriched solid precipitate portion and acarotenoid-depleted liquid portion, and (c) separating thecarotenoid-enriched solid portion from the carotenoid-depleted liquidportion.
 22. The process of claim 21 wherein the liquid fraction iscontacted with about 2 to about 4 wt % calcium lactate.
 23. The processof claim 22 wherein the liquid fraction is contacted with the calciumlactate for a time period of less than about 10 minutes.
 24. A processfor extracting carotenoids from a carotenoid-containing natural sourcewhich comprises the steps of:(a) separating a natural source of acarotenoid into a carotenoid-containing liquid fraction and a pulpfraction, (b) contacting the carotenoid-containing liquid fraction withan effective fractionating amount of calcium gluconate so as tofractionate the liquid fraction into a carotenoid-enriched solidprecipitate portion and a carotenoid-depleted liquid portion, and (c)separating the carotenoid-enriched solid portion from thecarotenoid-depleted liquid portion.
 25. The process of claim 24 whereinthe liquid fraction is contacted with about 4 to about 6 wt % calciumgluconate.
 26. The process of claim 25 wherein the liquid fraction iscontacted with the calcium gluconate for a time period of less thanabout 10 minutes.